Vitamin D and calcium deficiencies are risk factors for age-related bone loss and increased fracture risk. Decreased intestinal calcium absorption (which correlates with decreased expression of TRPV6 and calbindin-D9k, the two major molecular targets of 1,25(OH)2D3 in the intestine) combined with an increase in the catabolism of 1,25(OH)2D3 by 24(OH)ase contribute to age related bone loss. Although a principal action of 1,25(OH)2D3 is intestinal calcium absorption, we are far from understanding the mechanisms involved in vitamin D induced transport of calcium. In addition, the mechanisms by which inadequate vitamin D status contributes to osteoporosis are not yet known. The unifying hypothesis of this exploratory study is that basic mechanisms involved in 1,25(OH)2D3 mediated intestinal calcium absorption remain to be defined, that a complex of cofactors, including those associated with epigenetic regulation, are involved in vitamin D receptor (VDR) mediated events involved in maintaining calcium homeostasis and that there is a deterioration in this process with aging. To test this hypothesis in the first specific aim we propose to examine the functional interrelationship between calbindin and TRPV6. In initial studies using double KO mice we found that intestinal calcium absorption in response to low calcium or 1,25(OH)2D3 is least efficient in the absence of both calbindin-D9k and TRPV6 (compared to WT and single KO mice), suggesting that TRPV6 and calbindin-D9k (which are co-localized in the intestine) act together to affect calcium absorption. We hypothesize that calbindin-D9k is not a facilitator of calcium diffusion (thus challenging the traditional model of vitamin D mediated transcellular calcium absorption) but rather a modulator of calcium influx via TRPV6. Studies are proposed (using confocal microscopy, co-ip., GST pull down assays and electrophysiology) that will enable us to understand for the first time the functional interrelationship between calbindin-D9k and TRPV6 and thus their role in 1,25(OH)2D3 regulated intestinal calcium transport. Findings from these studies may lead to the development of pharmacological tools to enhance intestinal calcium absorption and thus help to maintain calcium responsiveness during aging. In specific aim 2 we propose to examine mechanisms by which 1,25(OH)2D3 acts to maintain calcium homeostasis and changes in vitamin D regulation that occur with aging . Besides the functional significance of target proteins it is also important to understand the molecular mechanisms by which 1,25(OH)2D3 controls calcium homeostasis. Epigenomic control is a new and rapidly evolving field and my lab has obtained the first evidence that specific protein methyltransferases cooperate with the p160 coactivator GRIP1 in regulating 1,25(OH)2D3 target gene expression. We will examine the role of CARM1 methyltransferase and cooperating cofactors in the regulation of key 1,25(OH)2D3 target genes including TRPV6 and 24(OH)ase and how the function of VDR, VDR associated factors and epigenetic regulation of VDR function are altered with age. In addition we will analyze by ChIP seq genome wide changes in histone methylation patterns and co-occurrence of regulatory cofactors in response to 1,25(OH)2D3 in VDR regulated genes (novel as well as known) in kidney and intestine and changes that occur with aging. VDR coactivators are master regulators of 1,25(OH)2D3 action. Understanding altered recruitment by 1,25(OH)2D3 of VDR and coactivators and epigenetic changes may be the key to understanding dysregulation of calcium homeostasis and altered responsiveness to vitamin D that occurs with aging. The long term goal is to define molecular pathways of vitamin D action in order to reveal new therapeutic strategies to sustain calcium balance. Identifying the most effective vitamin D analog to prevent or reverse deterioration of calcium homeostasis through transcriptional and epigenetic mechanisms could have long range implications for how bone diseases, particularly osteoporosis and osteopenia, are treated. This application is appropriate for an R21 since it involves considerable risk but may lead to a breakthrough that could have a major impact on a field of biomedical or clinical research.